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Anesthetic Preconditioning Inhibits Isoflurane-Mediated Apoptosis in the Developing Rat Brain

Peng, Jun, MD; Drobish, Julie K., MD; Liang, Ge, MD; Wu, Zhen, MD; Liu, Chunxia, MD; Joseph, Donald J., PhD; Abdou, Hossam, BS; Eckenhoff, Maryellen F., PhD; Wei, Huafeng, MD, PhD

doi: 10.1213/ANE.0000000000000380
Neuroscience in Anesthesiology and Perioperative Medicine: Research Report
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BACKGROUND: We hypothesized that preconditioning (PC) with a short exposure to isoflurane (ISO) would reduce neurodegeneration induced by prolonged exposure to ISO in neonatal rats, as previously shown in neuronal cell culture.

METHODS: We randomly divided 7-day-old Sprague-Dawley rats into 3 groups: control, 1.5% ISO, and PC + 1.5% ISO. The control group was exposed to carrier gas (30% oxygen balanced in nitrogen) for 30 minutes and then to carrier gas again for 6 hours the following day. The 1.5% ISO group was exposed to carrier gas for 30 minutes and then to 1.5% ISO for 6 hours the following day. The PC + 1.5% ISO group was preconditioned with a 30-minute 1.5% ISO exposure and then exposed to 1.5% ISO for 6 hours the following day. Blood and brain samples were collected 2 hours after the exposures for determination of neurodegenerative biomarkers, including caspase-3, S100β, caspase-12, and an autophagy biomarker Beclin-1.

RESULTS: Prolonged exposure to ISO significantly increased cleaved caspase-3 expression in the cerebral cortex of 7-day-old rats compared with the group preconditioned with ISO and the controls using Western blot assays. However, significant differences were not detected for other markers of neuronal injury.

CONCLUSIONS: The ISO-mediated increase in cleaved caspase-3 in the postnatal day 7 rat brain is ameliorated by PC with a brief anesthetic exposure, and differences were not detected in other markers of neuronal injury.

Published ahead of print August 5, 2014.

From the Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Jun Peng, MD, is currently affiliated with Department of Anesthesia, Second Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.

Zhen Wu, MD, is currently affiliated with Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Chunxia Liu, MD, is currently affiliated with Department of Anesthesiology, China-Japan Friendship Hospital, Beijing, China.

Accepted for publication June 13, 2014.

Published ahead of print August 5, 2014.

Funding: Supported by National Institute of General Medicine (NIGMS), National Institutes of Health (GM-073224, GM084979, GM084979-02S1 to HW), Bethesda, MD, March of Dimes Birth Defects Foundation Research Grant (#12-FY08-167 to HW), White Plains, NY, Research Fund at the Department of Anesthesiology and Critical Care, University of Pennsylvania (to HW), Philadelphia, PA.

The authors declare no conflicts of interest.

This report was previously presented, in part, at the Society for Neuroscience, 2010, San Diego, CA. Program #157.6.

Reprints will not be available from the authors.

Address correspondence to Huafeng Wei, MD, PhD, Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, 305 John Morgan Bldg, 3620 Hamilton Walk, Philadelphia, PA 19104. Address e-mail to Huafeng.Wei@uphs.upenn.edu.

Isoflurane (ISO) is a widely used general anesthetic for both adult and pediatric surgeries. Many studies have been performed to elucidate the harms and benefits of ISO on neurons1–9 and in the developing brain.4,10–12 Depending on the circumstances, ISO has been reported to have both neurotoxic and neuroprotective effects.

A large number of in vivo studies have shown that ISO causes apoptosis in the developing brain of various species of animals4,10,11,13–15 and that subsequent learning and memory are impaired.10,11 Furthermore, ISO has also been shown to be toxic in various cell culture models.2,4,16,17

Other studies have also shown that ISO can have neuroprotective effects. In particular, when ISO is used as a preconditioning drug, it can provide neuroprotection against various hypoxic-ischemic insults to the developing rodent brain.12,18–21 Previous in vitro work from our laboratory, and others, has shown that preconditioning with ISO has a protective effect on neuronal cell cultures subsequently exposed to ISO for a longer duration.2 However, inhibition of ISO-induced neuronal apoptosis during brain development by preconditioning has not yet been examined.

Given that ISO has been a successful preconditioning drug against subsequent anesthetic exposure for developing neurons in vitro2 and also against brain infarction induced by hypoxia and/or ischemia in vivo,12,18,20,22,23 we hypothesized that preconditioning with a short exposure to ISO would reduce neurodegeneration induced by a prolonged exposure to ISO in an animal model. We assessed the effects of 1.5% ISO exposure for 6 hours on apoptotic biomarkers in 7-day-old rats and then determined whether preconditioning with a short exposure to 1.5% ISO for 30 minutes changed the apoptotic response.

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METHODS

Animals

The experimental procedures and protocols used in this study were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. All efforts were made to minimize the number of animals used and their suffering. Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were housed with a 12-hour light–dark cycle at 22°C, with food and water provided ad libitum. Thirty-eight postnatal day 7 (P7) rats were used for the enzyme-linked immunosorbent assay (ELISA) and Western blots and 11 for immunohistochemistry, with approximately equal numbers of male and female rat pups randomly assigned to each condition.

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Anesthesia Exposure

The groups of rats were exposed to treatments in parallel. The minimum number of rats in each group was determined by a power analysis. We anticipated that a large effect size would be clinically significant and chose an effect size of 1.3, and using the desired statistical level of 0.8 and probability level of 0.05, we determined a minimum sample size per group (2-tailed hypothesis) of 11 animals. P7 rats were placed in plexiglass chambers resting in a 37°C water bath to maintain a constant environmental temperature. The rat pups were exposed in these chambers to carrier gas (30% oxygen balanced in nitrogen) for 30 minutes and then to 1.5% ISO for 6 hours the following day (1.5% ISO) or preconditioned (PC) with a 30-minute 1.5% ISO exposure and then exposed to 1.5% ISO for 6 hours the following day (PC + 1.5% ISO). The control animals were exposed to carrier gas (30% oxygen balanced in nitrogen) for 30 minutes and then to carrier gas again for 6 hours the following day in the plexiglass chambers but not in the water bath. Exposure to ISO for 30 minutes alone at P7 has been shown not to be detrimental,12 and thus this control group was not included. To maintain a steady state of anesthetic gas and to prevent accumulation of expired carbon dioxide within the chamber, we used 6 L of total gas flow throughout the experiments. The ISO, oxygen, and carbon dioxide levels in the chamber were monitored using IR absorbance (Ohmeda 5330, Datex-Ohmeda, Louisville, CO) as described in our previous studies.4,15,24 Two rats died during exposure to 1.5% ISO for 6 hours, 1 from the ISO alone group and the other from the PC plus ISO group.

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Determination of Plasma S100β

Two hours after the completion of the anesthetic treatment, P7 rats from the control, 1.5% ISO, and PC + 1.5% ISO groups were deeply anesthetized with 2% to 3% ISO. Blood (0.1 mL) was collected from the left ventricle and centrifuged to separate the plasma. We measured levels of S100β, a neuronal injury marker, using Sangtec 100 ELISA kits (DiaSorin Inc., Stillwater, MN) following the manufacturer’s protocol and as we described previously.25 Briefly, 50 μL plasma from each rat was placed in each well of a 96-well plate and mixed with 150 μL tracer from the kit and incubated for 2 hours. Afterward, 3,3′,5,5′tetramethylbenzidine substrate and stop solution were added to each well. The optical density was read at 450 nm. The sensitivity was determined by plotting the standard curve and then measuring concentrations of the samples from the standard curve.

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Western Blot Assays

Western blots were performed as we described previously.15,24 Two hours after the ISO exposure, after the mice were anesthetized and blood samples collected from the heart (see above), the mice were perfused with ice-cold saline through the heart and the parietal cortex dissected, frozen in liquid nitrogen, and stored at −80°C. At the time of the assay, the brain tissue from the P7 rat cortical tissue was thawed and homogenized and the total protein concentrations were quantified. The proteins were then separated by 12% gel electrophoresis and were transferred to a nitrocellulose membrane. The blots were incubated with an antibody against cleaved caspase-3 (Cell Signaling #9664; Cell Signaling Technology, Danvers, MA), caspase-12 (Cell Signaling #2202), or Beclin-1 (Cell Signaling #3495). The density was measured by Quantity One software (BIO-RAD version 4.5.0, BIO-RAD, Hercules, CA) and GS-800 Densitometer (BIO-RAD), and the data are expressed as the percent of control of the means from 1 blot per animal per group.

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Immunohistochemistry

Immunohistochemical localization of caspase-3 was performed in a separate group of P7 rats, as previously described.15 Briefly, 2 hours after the ISO exposure, P7 pups were deeply anesthetized with ISO and transcardially perfused with ice-cold saline before the brains were removed, fixed with 4% paraformaldehyde, cryoprotected in 30% sucrose, frozen in isopentane, and stored at −80°C. Coronal cryosections (10 μm) were incubated in 3% hydrogen peroxide, 10% normal goat serum, and cleaved caspase-3 antibody (1:400; Cell Signaling #9664) overnight at room temperature. The next day, the sections were incubated with Alexa Fluor® 594 goat anti-rabbit IgG (Life Technologies, Grand Island, NY) and coverslipped using ProLong® Gold Antifade Reagent containing the nuclear stain, 4’,6-diamidino-2-phenylindole (Invitrogen, Life Technologies). Quantitative imaging was conducted on an Olympus IX70 microscope equipped with a Cooke SensiCam camera (Applied Scientific Instrumentation, Eugene, OR) and IP lab 4.0 software (Biovision Technologies, Exton, PA). Caspase-positive and total number of cells were counted in the CA1 region of the hippocampus and the adjacent parietal cortex at ×20 magnification. The brain sampled and analyzed in parietal cortex was the same region used in the Western blot from the opposite brain hemisphere. The mean number of cells was calculated from 3 sections per animal and the data expressed as the percentage of caspase-3–positive cells in each region.

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Statistical Analysis

All data were analyzed using the Mann-Whitney U test to determine between-group differences and exact P values using STATA statistical software (StataCorp LP, College Station, TX). Differences were considered statistically significant at P < 0.01.

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Results

Preconditioning Significantly Reduces ISO’s Apoptotic Effect

This study tested the effects of PC, with a short exposure to ISO before a prolonged exposure to ISO, on apoptotic neurodegeneration in postnatal rats. The level of apoptosis was evaluated by determining cleaved caspase-3 levels in the cerebral cortex using Western blot immunoassays (Fig. 1A). The cleaved caspase-3 levels, expressed as percent of control, in the P7 rats exposed to ISO alone were significantly higher than the PC group (P = 0.0009) or the controls (P = 0.0004; Fig. 1B; Tables 1 and 2). In addition, the animals with PC with ISO were also significantly different from controls (P = 0.0007; Fig. 1B; Tables 1 and 2). When the P7 cortex and hippocampus were quantitatively analyzed for the immunohistochemical localization of caspase-3 (Fig. 2), significant differences were not detected in the number of caspase-3–positive cells in the ISO-exposed group compared with controls or the PC group in the cortex or the hippocampus (Fig. 2, B and C; Tables 1 and 2). These data support the claim that PC, with a short exposure to ISO, before a prolonged ISO exposure, significantly reduces the ISO-mediated apoptotic neurodegeneration in the developing rat brain using Western blot assays.

Figure 1

Figure 1

Table 1

Table 1

Table 2

Table 2

Figure 2

Figure 2

The level of neurodegeneration was further studied by determining the plasma levels of S100β, a marker of neuronal injury, in P7 rats exposed to 1.5% ISO for 6 hours, with and without ISO PC (Fig. 3). The ELISA assay did not reveal significant differences in plasma S100β levels between these groups (Tables 1 and 2).

Figure 3

Figure 3

The effect of PC on the apoptotic pathway was also examined by caspase-12 activation in the developing brain. Western blot analysis of caspase-12 levels in the P7 cerebral cortex (Fig. 4A) after either a 6-hour exposure to 1.5% ISO or PC with ISO before the prolonged exposure were not significantly different from controls (Fig. 4B; Tables 1 and 2).

Figure 4

Figure 4

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Effect of ISO Treatment on Autophagy

Autophagy, after anesthetic exposures, with and without PC, was examined in the P7 developing brain by measuring Beclin-1 levels. Western blot analysis (Fig. 5A) showed that a 6-hour ISO exposure did not significantly change Beclin-1 levels (Fig. 5B; Tables 1 and 2).

Figure 5

Figure 5

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DISCUSSION

This study provides new evidence that PC with ISO, before a long ISO exposure, significantly decreases ISO-mediated apoptosis in the developing brain. This finding is based on a significant reduction in the caspase-3 levels using Western blot assays. However, significant differences were not detected in caspase-3 levels in the cerebral cortex and hippocampus after either ISO exposure or PC. Other markers of neuronal injury, S100β, caspase-12, and Beclin-1, were not significantly affected by either the prolonged ISO exposure or PC. While we have been able to exclude a large effect of these markers, it is possible that we could not detect significant effects due to our small sample size (e.g., Figs. 2 and 3).

Caspase-3 is one of the final mediators of the apoptotic pathway and is a well-established biomarker of apoptosis. The most important aspect of our results is that ISO PC decreased caspase-3 levels induced by a prolonged ISO exposure in the developing brain. In a similar study, Shu et al26 showed that xenon pretreatment before a combined ISO/nitrous oxide exposure decreased apoptosis, while nitrous oxide PC had no effect. Furthermore, we have previously shown that sevoflurane PC can also inhibit neuronal cell death induced by prolonged exposure to ISO.2 Thus, more studies are necessary to determine the mechanism for the protective effect of PC so that novel approaches can be developed to mimic this effect.

Anesthetics have been shown to be both neuroprotective and neurotoxic in the developing brain, and thus concentrations and durations must be considered in pediatric anesthetic practice. Our previous study suggested that prolonged exposure to sevoflurane can induce neuronal damage in vitro.27 The IV anesthetic, propofol, has also been shown to be both neurotoxic28,29 and neuroprotective against brain damage induced by ischemia and other stress factors.30,31 Therefore, it is important to investigate the dose and time responses of anesthetic-induced effects in the developing brain to use their neuroprotective features but minimize their neurotoxic effects.

S100β, a dimeric cytosolic calcium-binding protein released by glial cells, is a biomarker of blood–brain barrier dysfunction32 and overall brain distress.33 It has been studied clinically as a biomarker for traumatic brain injury and hypoxic-ischemic brain injury.34,35 We have previously studied S100β in the developing fetal rat brain and found that an in utero exposure to 3% ISO for 1 hour resulted in higher levels of S100β in the plasma of fetal rats when compared with controls.25 Furthermore, we showed an increase in plasma S100β after exposure to a subclinical concentration of ISO in neonatal mice.15 Though the current study did not show a significant difference in S100β, S100β may be a useful biomarker to detect anesthetic-mediated damage in the developing brain as indicated above, although further studies are needed to investigate its role in pediatric patients.

Caspase-12, part of the apoptotic pathway, is activated by disruption of the calcium homeostasis in the endoplasmic reticulum (ER).36 Anesthetics have been shown to cause calcium dysregulation in the ER via multiple mechanisms.37 In immature hippocampal neurons, ISO exposure was shown to enhance γ-aminobutyric acid–induced intracellular calcium increase, which was blocked by dantrolene, indicating that ISO exposure causes ryanodine receptor–dependent calcium release from the ER,17 which is consistent with our previous studies in different types of neurons.5 Other studies also suggest that ISO exposure during brain development causes increased activation of inositol triphosphate receptors (InsP3R), resulting in increased calcium release from the ER leading to cell damage and neurodegeneration.3,4,27,38 Furthermore, caspase-12–positive neurons in the hippocampus were significantly increased in fetal rats exposed to 1.3% ISO for 4 hours.39 Caspase-12 has also been shown to indirectly activate caspase-3 in the neuronal apoptotic pathway.40 Although a previous study suggested that ISO-induced neuroapoptosis during brain development in rodents involved both intrinsic and extrinsic pathways,41 it is not clear whether the caspase-12–dependent pathway is also involved. In this study, we did not find significant caspase-12 activation after the ISO exposures, possibly because our ISO concentration may not have been high enough to cause ER stress and caspase-12–dependent neuroapoptosis. Further dose-dependent and exposure duration studies are needed to clarify this question.

Beclin-1, a protein required for autophagosome formation, is an important regulator and biomarker of autophagy activity.42–44 Interestingly, autophagy may have both beneficial and harmful effects on the brain, depending on the experimental conditions. Autophagy appears to be essential to both ischemic and hyperbaric oxygen PC before cerebral ischemia.45 Furthermore, reducing Beclin-1 levels has been shown to exacerbate neurodegeneration in Alzheimer disease models, while overexpression of Beclin-1 can prevent neuronal cell death.46 Autophagy activity can be regulated by InsP3R activity, while ISO has been shown to activate InsP3R and cause cell apoptosis by overactivation of InsP3R.47,48 In addition, autophagy activity may be an upstream regulator of apoptosis, and excessive autophagy may lead to cell death by apoptosis. The effects of InsP3R activity on autophagy depend on the level of InsP3R activation, which then determines whether the effect will be protective or toxic. While this study did not find an effect on Beclin-1 expression at P7 after exposure to 1 concentration of ISO, further studies are needed to investigate the effects of general anesthetics on cell autophagy, and therefore neuroprotection or neurotoxicity, especially in the developing brain.

While we have not yet discovered the mechanism by which ISO PC prevents ISO-induced apoptosis, other groups have studied ISO PC before cerebral ischemia and have linked several mechanisms to this process. One study found that ISO PC caused a decrease in glutamate receptor activation,49 and another found that it decreased protein aggregation.50 Changes in the expression of various genes have also been discovered, but the significance of these genetic changes has not been fully determined.51–53 Furthermore, ISO may provide PC neuroprotection by causing a moderate increase of cytosolic calcium concentrations via adequate activation of the InsP3R calcium channel.54,55 While we recognize that ISO PC for brain ischemia is not the same as for a subsequent anesthetic exposure, it is possible that there may be similarities in the cascade of events and consequences. One of the limitations of the current study is that we only investigated a few of the potential mechanisms underlying the dual effects of neuroprotection and neurotoxicity caused by ISO. Future experiments could continue to address the many other mechanisms that are likely involved in this process.

The long-term behavioral adaptations to the effects of preadolescent drug exposures are more permanent compared with the same exposures later in life,56 with the peak period of anesthetic-induced apoptosis occurring during synaptogenesis.57 This vulnerable period during rat development is between approximately postnatal days 2 and 14 and between postconception day 153 and postnatal day 288 in the human, based on a species prediction model recently developed to correlate the timing of neural events between species.58 Translating exact developmental milestones between rats and humans is complicated. Postnatal brain maturation in the rat encompasses many developmental events, such as neurogenesis, neuronal migration, synaptogenesis, and apoptosis, the extent of which varies greatly, depending on the brain region of interest.59,60

The present study tested the hypothesis that PC with ISO can prevent apoptosis caused by a prolonged exposure to ISO, and our results with caspase-3, but not other markers of neuronal injury, support this hypothesis and indicate that in vivo ISO PC is neuroprotective, while a prolonged exposure to ISO is neurotoxic during early postnatal brain development. It is important to determine the optimal concentration and duration range for anesthetic exposures during postnatal brain development, which has implications for our pediatric patients.

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DISCLOSURES

Name: Jun Peng, MD.

Contribution: This author helped design the study, conduct the study, and analyze the data.

Attestation: Jun Peng has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Julie K. Drobish, MD.

Contribution: This author helped with the writing of the manuscript.

Attestation: Julie K. Drobish has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Ge Liang, MD.

Contribution: This author helped design the study, conduct the study, and analyze the data.

Attestation: Ge Liang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Zhen Wu, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Zhen Wu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Chunxia Liu, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Chunxia Liu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Donald J. Joseph, PhD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Donald J. Joseph reviewed the analysis of the data and approved the final manuscript.

Name: Hossam Abdou, BS.

Contribution: This author helped with the writing of the manuscript.

Attestation: Hossam Abdou reviewed the analysis of the data and approved the final manuscript.

Name: Maryellen F. Eckenhoff, PhD.

Contribution: This author helped with the writing of the manuscript.

Attestation: Maryellen F. Eckenhoff reviewed the analysis of the data and approved the final manuscript.

Name: Huafeng Wei, MD, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Huafeng Wei has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

This manuscript was handled by: Gregory J. Crosby, MD.

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ACKNOWLEDGMENTS

The authors would like to thank Rebecca Speck, PhD, MPH, Department of Anesthesiology and Critical Care, University of Pennsylvania, for advice and assistance with the statistical analyses.

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REFERENCES

1. Liang G, Wang QJ, Li Y, Kang B, Eckenhoff MF, Eckenhoff RG, Wei H. A presenilin-1 mutation renders neurons vulnerable to isoflurane toxicity. Anesth Analg. 2008;106:492–500
2. Wei H, Liang G, Yang H. Isoflurane preconditioning inhibited isoflurane-induced neurotoxicity. Neurosci Lett. 2007;425:59–62
3. Wang Q, Liang G, Yang H, Wang S, Eckenhoff MF, Wei H. The common inhaled anesthetic isoflurane increases aggregation of huntingtin and alters calcium homeostasis in a cell model of Huntington’s disease. Toxicol Appl Pharmacol. 2011;250:291–8
4. Zhao Y, Liang G, Chen Q, Joseph DJ, Meng Q, Eckenhoff RG, Eckenhoff MF, Wei H. Anesthetic-induced neurodegeneration mediated via inositol 1,4,5-trisphosphate receptors. J Pharmacol Exp Ther. 2010;333:14–22
5. Wei H, Kang B, Wei W, Liang G, Meng QC, Li Y, Eckenhoff RG. Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently. Brain Res. 2005;1037:139–47
6. Xie Z, Dong Y, Maeda U, Moir RD, Xia W, Culley DJ, Crosby G, Tanzi RE. The inhalation anesthetic isoflurane induces a vicious cycle of apoptosis and amyloid beta-protein accumulation. J Neurosci. 2007;27:1247–54
7. Culley DJ, Boyd JD, Palanisamy A, Xie Z, Kojima K, Vacanti CA, Tanzi RE, Crosby G. Isoflurane decreases self-renewal capacity of rat cultured neural stem cells. Anesthesiology. 2011;115:754–63
8. Zheng S, Zuo Z. Isoflurane preconditioning reduces Purkinje cell death in an in vitro model of rat cerebellar ischemia. Neuroscience. 2003;118:99–106
9. Zuo Z, Wang Y, Huang Y. Isoflurane preconditioning protects human neuroblastoma SH-SY5Y cells against in vitro simulated ischemia-reperfusion through the activation of extracellular signal-regulated kinases pathway. Eur J Pharmacol. 2006;542:84–91
10. Ma D, Williamson P, Januszewski A, Nogaro MC, Hossain M, Ong LP, Shu Y, Franks NP, Maze M. Xenon mitigates isoflurane-induced neuronal apoptosis in the developing rodent brain. Anesthesiology. 2007;106:746–53
11. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci. 2003;23:876–82
12. Zhao P, Zuo Z. Isoflurane preconditioning induces neuroprotection that is inducible nitric oxide synthase-dependent in neonatal rats. Anesthesiology. 2004;101:695–703
13. Palanisamy A, Baxter MG, Keel PK, Xie Z, Crosby G, Culley DJ. Rats exposed to isoflurane in utero during early gestation are behaviorally abnormal as adults. Anesthesiology. 2011;114:521–8
14. Brambrink AM, Evers AS, Avidan MS, Farber NB, Smith DJ, Zhang X, Dissen GA, Creeley CE, Olney JW. Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology. 2010;112:834–41
15. Liang G, Ward C, Peng J, Zhao Y, Huang B, Wei H. Isoflurane causes greater neurodegeneration than an equivalent exposure of sevoflurane in the developing brain of neonatal mice. Anesthesiology. 2010;112:1325–34
16. Head BP, Patel HH, Niesman IR, Drummond JC, Roth DM, Patel PM. Inhibition of p75 neurotrophin receptor attenuates isoflurane-mediated neuronal apoptosis in the neonatal central nervous system. Anesthesiology. 2009;110:813–25
17. Zhao YL, Xiang Q, Shi QY, Li SY, Tan L, Wang JT, Jin XG, Luo AL. GABAergic excitotoxicity injury of the immature hippocampal pyramidal neurons’ exposure to isoflurane. Anesth Analg. 2011;113:1152–60
18. Sakai H, Sheng H, Yates RB, Ishida K, Pearlstein RD, Warner DS. Isoflurane provides long-term protection against focal cerebral ischemia in the rat. Anesthesiology. 2007;106:92–9
19. McAuliffe JJ, Joseph B, Vorhees CV. Isoflurane-delayed preconditioning reduces immediate mortality and improves striatal function in adult mice after neonatal hypoxia-ischemia. Anesth Analg. 2007;104:1066–77
20. Li L, Zuo Z. Isoflurane preconditioning improves short-term and long-term neurological outcome after focal brain ischemia in adult rats. Neuroscience. 2009;164:497–506
21. McAuliffe JJ, Loepke AW, Miles L, Joseph B, Hughes E, Vorhees CV. Desflurane, isoflurane, and sevoflurane provide limited neuroprotection against neonatal hypoxia-ischemia in a delayed preconditioning paradigm. Anesthesiology. 2009;111:533–46
22. Michenfelder JD, Sundt TM, Fode N, Sharbrough FW. Isoflurane when compared to enflurane and halothane decreases the frequency of cerebral ischemia during carotid endarterectomy. Anesthesiology. 1987;67:336–40
23. Zhou Y, Lekic T, Fathali N, Ostrowski RP, Martin RD, Tang J, Zhang JH. Isoflurane posttreatment reduces neonatal hypoxic-ischemic brain injury in rats by the sphingosine-1-phosphate/phosphatidylinositol-3-kinase/Akt pathway. Stroke. 2010;41:1521–7
24. Li Y, Liang G, Wang S, Meng Q, Wang Q, Wei H. Effects of fetal exposure to isoflurane on postnatal memory and learning in rats. Neuropharmacology. 2007;53:942–50
25. Wang S, Peretich K, Zhao Y, Liang G, Meng Q, Wei H. Anesthesia-induced neurodegeneration in fetal rat brains. Pediatr Res. 2009;66:435–40
26. Shu Y, Patel SM, Pac-Soo C, Fidalgo AR, Wan Y, Maze M, Ma D. Xenon pretreatment attenuates anesthetic-induced apoptosis in the developing brain in comparison with nitrous oxide and hypoxia. Anesthesiology. 2010;113:360–8
27. Yang H, Liang G, Hawkins BJ, Madesh M, Pierwola A, Wei H. Inhalational anesthetics induce cell damage by disruption of intracellular calcium homeostasis with different potencies. Anesthesiology. 2008;109:243–50
28. Cattano D, Young C, Straiko MM, Olney JW. Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain. Anesth Analg. 2008;106:1712–4
29. Tu S, Wang X, Yang F, Chen B, Wu S, He W, Yuan X, Zhang H, Chen P, Wei G. Propofol induces neuronal apoptosis in infant rat brain under hypoxic conditions. Brain Res Bull. 2011;86:29–35
30. Cai J, Hu Y, Li W, Li L, Li S, Zhang M, Li Q. The neuroprotective effect of propofol against brain ischemia mediated by the glutamatergic signaling pathway in rats. Neurochem Res. 2011;36:1724–31
31. Kawaguchi M, Furuya H, Patel PM. Neuroprotective effects of anesthetic agents. J Anesth. 2005;19:150–6
32. Cata JP, Abdelmalak B, Farag E. Neurological biomarkers in the perioperative period. Br J Anaesth. 2011;107:844–58
33. Michetti F, Gazzolo D. S100B protein in biological fluids: a tool for perinatal medicine. Clin Chem. 2002;48:2097–104
34. Bloomfield SM, McKinney J, Smith L, Brisman J. Reliability of S100B in predicting severity of central nervous system injury. Neurocrit Care. 2007;6:121–38
35. Tavarez MM, Atabaki SM, Teach SJ. Acute evaluation of pediatric patients with minor traumatic brain injury. Curr Opin Pediatr. 2012;24:307–13
36. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, Yuan J. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature. 2000;403:98–103
37. Wei H. The role of calcium dysregulation in anesthetic-mediated neurotoxicity. Anesth Analg. 2011;113:972–4
38. Wei H, Liang G, Yang H, Wang Q, Hawkins B, Madesh M, Wang S, Eckenhoff RG. The common inhalational anesthetic isoflurane induces apoptosis via activation of inositol 1,4,5-trisphosphate receptors. Anesthesiology. 2008;108:251–60
39. Kong F, Xu L, He D, Zhang X, Lu H. Effects of gestational isoflurane exposure on postnatal memory and learning in rats. Eur J Pharmacol. 2011;670:168–74
40. Hitomi J, Katayama T, Taniguchi M, Honda A, Imaizumi K, Tohyama M. Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12. Neurosci Lett. 2004;357:127–30
41. Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V. Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience. 2005;135:815–27
42. Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011;18:571–80
43. Vicencio JM, Ortiz C, Criollo A, Jones AW, Kepp O, Galluzzi L, Joza N, Vitale I, Morselli E, Tailler M, Castedo M, Maiuri MC, Molgó J, Szabadkai G, Lavandero S, Kroemer G. The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1. Cell Death Differ. 2009;16:1006–17
44. Maiuri MC, Criollo A, Kroemer G. Crosstalk between apoptosis and autophagy within the Beclin 1 interactome. EMBO J. 2010;29:515–6
45. Wei K, Wang P, Miao CY. A double-edged sword with therapeutic potential: an updated role of autophagy in ischemic cerebral injury. CNS Neurosci Ther. 2012;18:879–86
46. Jaeger PA, Wyss-Coray T. Beclin 1 complex in autophagy and Alzheimer disease. Arch Neurol. 2010;67:1181–4
47. Høyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, Bianchi K, Fehrenbacher N, Elling F, Rizzuto R, Mathiasen IS, Jäättelä M. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell. 2007;25:193–205
48. Høyer-Hansen M, Jäättelä M. Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium. Cell Death Differ. 2007;14:1576–82
49. Zheng S, Zuo Z. Isoflurane preconditioning decreases glutamate receptor overactivation-induced Purkinje neuronal injury in rat cerebellar slices. Brain Res. 2005;1054:143–51
50. Zhang HP, Yuan LB, Zhao RN, Tong L, Ma R, Dong HL, Xiong L. Isoflurane preconditioning induces neuroprotection by attenuating ubiquitin-conjugated protein aggregation in a mouse model of transient global cerebral ischemia. Anesth Analg. 2010;111:506–14
51. Kitano H, Kirsch JR, Hurn PD, Murphy SJ. Inhalational anesthetics as neuroprotectants or chemical preconditioning agents in ischemic brain. J Cereb Blood Flow Metab. 2007;27:1108–28
52. Zhao P, Peng L, Li L, Xu X, Zuo Z. Isoflurane preconditioning improves long-term neurologic outcome after hypoxic-ischemic brain injury in neonatal rats. Anesthesiology. 2007;107:963–70
53. Zhu W, Wang L, Zhang L, Palmateer JM, Libal NL, Hurn PD, Herson PS, Murphy SJ. Isoflurane preconditioning neuroprotection in experimental focal stroke is androgen-dependent in male mice. Neuroscience. 2010;169:758–69
54. Bickler PE, Fahlman CS. The inhaled anesthetic, isoflurane, enhances Ca2+-dependent survival signaling in cortical neurons and modulates MAP kinases, apoptosis proteins and transcription factors during hypoxia. Anesth Analg. 2006;103:419–29
55. Gray JJ, Bickler PE, Fahlman CS, Zhan X, Schuyler JA. Isoflurane neuroprotection in hypoxic hippocampal slice cultures involves increases in intracellular Ca2+ and mitogen-activated protein kinases. Anesthesiology. 2005;102:606–15
56. Andersen SL. Trajectories of brain development: point of vulnerability or window of opportunity? Neurosci Biobehav Rev. 2003;27:3–18
57. Wang C, Slikker W Jr.. Strategies and experimental models for evaluating anesthetics: effects on the developing nervous system. Anesth Analg. 2008;106:1643–58
58. Workman AD, Charvet CJ, Clancy B, Darlington RB, Finlay BL. Modeling transformations of neurodevelopmental sequences across mammalian species. J Neurosci. 2013;33:7368–83
59. Pressler R, Auvin S. Comparison of brain maturation among species: an example in translational research suggesting the possible use of bumetanide in newborn. Front Neurol. 2013;4:36
60. Clancy B, Finlay BL, Darlington RB, Anand KJ. Extrapolating brain development from experimental species to humans. Neurotoxicology. 2007;28:931–7
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